Aim 1: Enhancement of the Microscope and its Computer Control The objective of this core TR&D project is to develop and improve EM techniques for image contrast enhancement and 3D information extraction from biological specimens, particularly thick sections, with computer-controlled specimen positioning, electron optics and a use of a direct digital readout image acquisition system being developed under a separate subproject (see following project description). Progress has been made in the following areas: Computer control and microscope automation: A microscope control library has been established and new routines are added as need arises. All optical parameters and most mechanical controls (such as the four axes of the stage) can now be remotely controlled by a local host as well as by workstations linked to the host by the computer network. Semi-automated tomography data acquisition software has been developed and used successfully for film-based and slow-scan CCD camera-based tomography. In addition, we have implemented an automated specimen survey capability utilizing stage movement. A large mosaic of 10 x 10 or more 1k x 1k images can be collected automatically, covering a few square mm. This montage is sent to the remote user before a telemicroscopy session and serves as a low magnification map for selecting potentially interesting regions to explore at higher magnifications. Characterization of the high tilt coil-enabled optical sectioning possibilities with IVEM: During the previous year JEOL finally completed the installation of a new set of image shift coils above the mini-lens assembly, below the objective lens. Delivery of these long-awaited coils allowed us to begin to test the final part of our scheme to develop electron optical sectioning by creating a digital rotary hollow-cone illumination above the specimen.. Briefly we first developed a unique electron optical design for this IVEM which enabled operation in the so-called "B" mode. This new optical scheme has several advantages, some of which were noted above: [unreadable] It provides for high contrast, high resolution imaging of thick sections due to the removal of multiply scattered electrons and an energy filtering effect. [unreadable] It enables on-line 3D observation and stereo views may be displayed quickly with tilts on any azimuth as the method uses beam tilt instead of stage tilt; [unreadable] It provided us with a test-bed for experimentation toward the development of a fully functional "optical sectioning" electron microscope. This work involved the use of specially designed scan and descan coils to form a synchronized hollow-cone illumination to reduce depth of field. The new coils seem to be perfect and preliminary data demonstrating optical sectioning capabilities has now been obtained. Addition of confocal and 2-photon microscopes: Although not specifically described as a technology aim in the original NCMIR proposal to NCRR , the availability of advanced light microscopes are essential to the activities of the resource for performing correlative light and electron microscope 3D analyses. Recognizing this we arranged for BioRad and Nikon to become research partners with NCMIR and to donate upgrades or provide instruments for our use. During the current period of funding, we upgraded the older Biorad 600 laser-scanning confocal housed in the NCMIR (originally purchased in 1989 for specific research projects by Ellisman and Terry) to a new 24bit 1024 system. This was done through a generous partnering arrangement with BioRad at no cost to the Resource. In addition, Nikon Corporation (Japan) established a research agreement (funded by a grant to Ellisman and UCSD) which included a donation of a Nikon RCM8000 video-rate confocal system . This system has now been modified by us to provide both UV or 3 channel visible light high speed confocal imaging as well a high speed multi-photon imaging. The microscope, incorporates a femto-second, tunable, pulsed laser providing excitation at wavelengths from 690 to 1050 nm, an NCMIR designed and constructed pre-chirper optical system for laser pulse-width compression, and a non-confocal detection assembly. A 75% increase in fluorescent emission is consistently obtained with the use of the prechirper optics. The non-confocal assembly is capable of detecting the ratio of two emission wavelengths and provides a 125% increase in detection efficiency compared to confocal detection. Ratio imaging and optical sectioning can therefore be performed more efficiently using multiphoton imaging than with confocal optics. Useful two-photon images can be acquired at video rate with a laser power as low as 2.7 mW at the specimen using genetically modified green fluorescent proteins (GFPs). To our knowledge, this is the first system to deliver video rate (and faster) 2-photon images of living preparations. We recently succeeded in acquiring images of moving GFP-labeled bacteria at rates of 1 frame/4msec. (32x512 pixels). This instrument will be further modified for use in at least one of the correlated microscopy projects with David Kleinfeld's group proposed in our pending NCRR renewal application.